FT-0554 substance and its production

ABSTRACT

The present invention is to obtain novel FT-0554 substance which is useful for treatment of helminthiasis. The present invention comprising the steps of culturing the microorganism belonging to fungi having producing activity of FT-0554 substance represented by the following formula [I] 
                 
 
     subjected to accumulation of FT-0554 substance in the cultured medium, and isolating FT-0554 substance from the said cultured mass. The medicament useful for treatment of parasitic infection, specifically helminthiasis can be obtained.

FIELD OF THE INVENTION

[0001] This invention relates to novel FT-0554 substance useful fortreatment for infection of parasite, especially helminth, and itsproduction.

PRIOR ARTS

[0002] Parasitosis is reducing as a result of improvement in sanitaryconditions and progress of anthelmintics. Recently, however, the importparasitosis, zoonotic parasitosis, opportunistic parasitosis andparasitosis originated from perishable foods are prevailing and becomecrucial problems, Further the parasitosis produces large economicalburdens in the stock-farming and agriculture. For infection of helminthin the parasite, at present, avermectins, mebendazole, praziquantel, andothers are used for treatment of helminth.

PROBLEMS TO BE SOLVED BY THE INVENTION

[0003] Anthelmintics used at present, such as avermectins, mebendazoleand praziquantel, are not always sufficient for satisfactory inusefulness and toxicity, and the anthelmintics, which can solve theseproblems, are strongly, required.

[0004] Consequently, the present invention provides novel FT-0554substance, which can satisfy the above requirements, and its production.

MEANS FOR SOLVING THE PROBLEMS

[0005] We have studided NADH-fumarate reductase, which was one of thepromising targets against anthelmintics, in the electron transportsystem of the helminth, and explored for screening in the microbialculture. We have found that novel FT-0554 substance had NADH-fumaratereductase inhibitory activity, and completed the present inventionaccording to this acknowledge.

[0006] An object of the present invention is to provide FT-0554substance shown by the compound of the formula [1]

[0007] Another object of the present invention is to provide a processfor production of FT-0554 substance of the formula [1]

[0008] which comprises culturing a microorganism belonging to fungushaving FT-0554 substance producing activity, accumulating FT-0554substance in the cultured medium and isolating FT-0554 substance fromthe cultured medium.

[0009] Further object of the present invention is to provide amicroorganism belonging to fungi and having FT-0554 substance producingactivity of the above process being Aspergillus niger FT-0554. Stillfurther object of the present invention is to provide a microorganismbelonging to fungi and having FT-0554 substance producing activity beingAspergillus niger FT-0554.

[0010] FT-0554 substance producing microorganism is the fungi havingFT-0554 substance producing activity and is not limited. Preferableexample of a microorganism used for production of FT-0554 is a fungusstrain FT-0554 isolated from a newly collected sponge by the inventorsof the present invention.

[0011] Taxonomical properties of the microorganism are illustrated asfollows.

[0012] Taxonomical properties of a strain FT-0554.

[0013] (1) Cultured properties on various media

[0014] Results of macroscopic observation of the strain of the presentmicroorganism cultured at 25° C. for 7 days are shown in Table 1. TABLE1 Growth condition Color of Color of on the medium surface of reverseside soluble Medium (diameter of colony) colony of colony pigmentCzapek-yeast good (82 mm) black brown pale yellow none extract agarvelvety, entire Malt extract good (81 mm) black brown pale yellow noneagar medium velvety, entire 20% sucrose Czapek-yeast good (82 mm) blackbrown pale yellow none extract agar medium velvety, entirePotate-glucose good (>85 mm) black brown pale yellow none agar mediumvelvety, entire Miura agar medium moderate (40 mm) black brown whitenone velvety, entire

[0015] (2) Morphological properties

[0016] The microorganism of the present invention shows good growth onCzapek-yeast extract agar medium which contains seawater 50% (saltcontent 3.4%), malt extract agar medium, Czapek-yeast extract agarmedium which contains sucrose 20% and potato-glucose agar medium, withabundance of conidia.

[0017] Microscopical observation of colonies grown on Czapek-yeastextract agar medium shows transparent hyphae with septa, straight grownconidiophore on the substrate mycelia with length 500 μm-2.5 mm, andfoot-cell in the basement. Tops of conidiophores are hypertrophic fromspherical to subspherical with forming vesicles of diameter 35-60 μm.

[0018] Plural aspergillae consist of metulae and phialides with the sizeof 8.4-11.4×2.4-3.4 μm and 5.4-8.6×2.8-3.3 μm. respectively. Whole ofthe vesicles is covered with metulae with forming conidial headssegmented from spherical to cylindrical. Conidia is globose with a sizeof diameter 3-4.5 μm having smooth to rough surface.

[0019] (3) Physiological properties

[0020] 1) Optimum growth condition

[0021] Optimum growth condition of the present strain is pH 5-7,temperature 16-36° C. and seawater concentration ¹⁾50-100%.

[0022]¹⁾: salt concentration 3.4% natural seawater is used

[0023] 2) Growth condition

[0024] Growth range of the strain is pH 3-10, temperature 12-45° C. andseawater concentration ²⁾ 0-100%.

[0025]²⁾: salt concentration 3.4% natural seawater is used

[0026] 3) Nature

[0027] Aerobic

[0028] As shown in the above, culture condition, taxonomical propertiesand physiological properties of the present microorganism strain FT-0554are compared with the known microorganism strains. The present strain isidentified as belonging to Aspergillus niger and referred to Aspergillusniger FT-0554.

[0029] The present microorganism strain has deposited as Aspergillusniger FT-0554 FERM P-16399 in National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Science and Technology, 1-3, Higashi 1 - chose,Tsukuba-shi, Ibaraki-ken, Japan on Sep. 1, 1997. Further, the presentmicroorganism strain was transferred to the microorganism depositionunder Budapest Treaty in National Institute of Bioscience andHuman-Technology, Agency of Industrial Science and Technology, Ministryof International Science and Technology, 1-3, Higashi 1-chome,Tsukuba-shi, Ibaraki-ken, Japan on Jul. 31, 1998, and was givendeposition No. FERM BP-6443 from the International Deposition Authority.

[0030] Production of FT-0554 substance of the present invention can beperformed by culturing microorganism belonging to fungi having producingactivity of FT-0554 substance and isolating from the cultured mass andpurifying the product. The microorganism strain used in the presentinvention can be the above microorganism strain, its variants andmutants, and all strains having FT-0554 substance producing activitybelonging to fungi.

[0031] Nutritional sources for production of the above FT-0554 substancecan be a nutritional source for fungi. Examples of nitrogen sources arecommercially available peptone, meat extract, corn steep liquor,cottonseed powder, peanut powder, soybean flour, yeast extract,NZ-amine, casein hydrolyzate, sodium nitrate, ammonium nitrate andammonium sulfate. Example of carbon sources are carbohydrate such asglycerin, starch, glucose, galactose and mannose, or carbon source suchas fats and oil, and inorganic salts such as sodium chloride, phosphate,calcium carbonate and magnesium sulfate. These can be used with orcombination thereof.

[0032] Trace metallic salt and animal, vegetable or mineral oil asantiform agent can be added if necessary. These are substance, which canbe assimilated by the producing strain and are useful for production ofFT-0554 substance, can be used. All the known medium for culturing thefungus can be used. Mass production of FT-0554 substance can preferablybe performed by a liquid culture. Culturing temperature can be appliedwithin the range of growing the producing microorganism strain andproducing FT-0554 substance. Culturing can be performed by selectingsuitable conditions depending on the nature of FT-0554 substanceproducing strain.

[0033] FT-0554 substance can be extracted by water immiscible organicsolvent such as chloroform and ethyl acetate from the culture liquid. Inaddition to the above extraction method, known isolation method used forlipophilic substance, for example adsorption chromatography, gelfiltration chromatography, scratching from thin layer chromatography,centrifugal counter current chromatography, HPLC, and the like with orwithout combination thereof or repeated operation, can be applied toobtain purified substance.

[0034] Physico-chemical properties of FT-0554 substance of the presentinvention are shown as follows.

[0035] (1) Nature white powder or amorphous

[0036] (2) Molecular weight 361.2374 (M+H, high resolution fast atombombardment mass spectroscopy)

[0037] (3) Molecular formula C₂₂H₃₂O₄

[0038] (4) Specific rotation: [a]_(D) ²⁵=+35.83 (c=0.1, 1-propanol)

[0039] (5) UV absorption maximum (in 1-propanol): As shown in FIG. 1,maximum absorption at 205 nm (shoulder, ε=10800) and 231 nm (ε=21000).

[0040] (6) IR absorption maximum (KBr Tab): As shown in FIG. 2, maximumabsorption at 3430, 2960, 2920, 2850, 1740, 1660, 1460, 1400, 1380,1180, 1120 and 1000 cm⁻¹

[0041] (7) ¹H-NMR: chemical shift in deuterochloroform (ppm) andspin-spin coupling constant (Hz) in Table 2

[0042] (8) ¹³C NMR: chemical shift in deuterochloroform (ppm) in Table 2

[0043] (9) Solubility in solvent: soluble in chloroform, ethanol,1-propanol, toluene and ethyl acetate, insoluble in water and n-hexane

[0044] (10) Color reaction: positive for sulfuric acid and iodine. TABLE2 ¹³C ¹H 170.6 s 145.2 d 5.79 dd (1 H, J = 6.9, 15.2) 138.9 d 5.46 dd (1H, J = 7.6, 15.2) 137.9 d 6.37 dd (1 H, J = 10.2, 15.2) 133.7 s 127.0 d5.76 d (1 H, J = 10.9 Hz) 126.0 d 6.02 dd (1 H, J = 10.2, 15.2) 124.6 d6.17 dd (1 H, J = 10.9, 15.2) 122.0 d 5.48 dd (1 H, J = 7.9, 15.2)  80.2d 4.92 d (1 H, J = 7.9)  68.0 d 4.57 s (1 H)  58.5 d 3.52 s (1 H)  58.2s  47.2 t 1.96 dd (1 H, J = 7.7, 13.7) 2.09 dd (1 H, J = 7.1, 13.7) 38.6 d 2.06 m (1 H)  34.8 d 2.42 m (1 H)  29.8 t 1.31 dq (2 H, J = 7.1,7.3)  20.2 q 0.98 d (3 H, J = 6.8)  19.4 q 0.97 d (3 H, J = 6.8)  17.8 q1.45 s (3 H)  16.5 q 1.69 s (3 H)  11.8 q 0.85 t (3 H, J = 7.3)

[0045] In Table 2, s: singlet, d: doublet, t: triplet, q: quartet, m:multiplet, H: number of proton, and J: spin-spin coupling constant (Hz).

[0046] As a result of detailed examination of physico-chemicalproperties and spectrum data of FT-0554 substance, FT-0554 substance isdetermined as the following chemical structure.

[0047] As shown in the above, physico-chemical properties of FT-0554substance are explained in detail, however no compound having identicalproperties have been reported. Consequently, FT-0554 substance isdefined as novel substance.

[0048] NADH-fumarate reductase inhibitory activity of FT-0554 of thepresent invention is explained as follows.

[0049] Muscles of Ascaris suum were homogenized in 120 mM sodiumphosphate solution (pH 7.0) and centrifuged at 3,000×g for 10 minutes tocollect the supernatant solution. The supernatant was furthercentrifuged at 10,000×g for 20 minutes to collect the precipitate. Theprecipitate was suspended in 120 mM sodium phosphate solution (pH 7.0)to obtain mitochondrial fraction.

[0050] After 10 μl of test sample dissolved in 50% dimethyl sulfoxidesolution was added into 96 holes microplate, 120 mM sodium phosphatesolution (pH 7.0) containing 0.35 mM NADH. 7.2 mM disodium fumarate and18 mg/ml bovine serum albumin was added thereto, and pre-incubated inthe microplate reader ELx808 (Bio-Tek industries Co.) at 37° C. for 5minutes.

[0051] Mitochondrial fraction of Ascaris suum 10 μl (protein content 0.3mg) was added therein and incubated at 37° C. for 10 minutes. Absorptionof NADH at 340 nm was measured every 15 seconds. As a result ofquantitative measurement of NADH-fumarate reductase activity shown bydecrease in the slope of absorbancy at 340 nm, 50% inhibition ofNADH-fumarate reductase activity were obtained at 2.8 μM of FT-0554substance. Consequently, FT-0554 substance can be expected to use asdrug for treatment or prevention of helminthiasis.

[0052] Antimicrobial activity of FT-0554 substance of the presentinvention is as follows.

[0053] Chloroform solution of the compound of the present invention (1mg/ml) 10 μl is dipped on a filter paper disk (Advantec Co. diameter 6mm). which is air-dried to remove the solvent. The air-dried to removethe solvent. The dried disks are put on the agar plates containing testorganisms, incubated at 37° C. or 27° C. for 24 hours, and diameter ofthe inhibition zone around the paper disk is measured. Result are shownin Table 3. TABLE 3 inhibition zone Test organisms diameter (mm)Escherichia coli KB213 (NIHJ) − Escherichia coli KB176 (NIHJ JC-2, IFO12734) − Pseudomonas aeruginosa P-3 KB105 + Xanthomonas oryzae KB88 −Micrococcus luteus KB40 (PCI 1001) − Staphylococcus aureus KB210 −Mycobacterium smegmatis KB42 (ATCC 607) − Bacillus subtilis KB27 (PCI219) − Bacteroides fragilis KB169 (ATCC 23745) − Acholeplasma laidrawiiKB174 − Candida albicans KF1 12 Saccharomyces cervisiae KF26 −Aspergillus niger KF103 (ATCC 6275) − Pyricularia oryzae KF180 ± Mucorracemosus KF223 (IFO 4581) 10

[0054] As shown in Table 3, FT-0554 substance of the present inventionhas weak growth inhibitory activity against some microorganisms.

BRIEF EXPLANATION OF THE DRAWINGS

[0055]FIG. 1 shows UV spectrum of FT-0554 substance of the presentinvention in 1-propanol solution (50 μM).

[0056]FIG. 2 shows IR spectrum of FT-0554 substance of the presentinvention (KBr Tab.).

EMBODIMENTS OF THE PRESENT INVENTION

[0057] The following examples illustrate the present invention, but isnot construed to limit the invention.

EXAMPLE

[0058] A loopful of Aspergillus niger FT-0554 (FERM BP-6443) cultured inagar slant medium was inoculated into the liquid medium (pH 7.0)containing glucose 2.0%, polypeptone (Nihon Seiyaku Co.) 0.5%, agar0.1%, yeast extract (Oriental Yeast Co.) 0.2%, magnesium sulfate 7hydrate 0.05% and pottassium dihydrogen phosphate 0.1% dissolved in 50%natural seawater (salt concentration 3.4% natural seawater was used) anddivided into 100 ml in a 500 ml Erlenmeyer flask, and shake cultured at27° C. for 2 days.

[0059] This seed culture liquid each 1 ml was inoculated into the liquidmedium containing potato dextrose broth (Difco Inc.) 2.4% dissolved in50% natural seawater (salt concentration 3.4% natural seawater was used)divided each 100 ml in a 500 ml Erlenmeyer flask (×30 flasks) and shakecultured at 27° C. for 96 hours.

[0060] Cultured liquid medium was centrifuged and obtained mycelia wasextracted with ethanol 3 lit. Ethanol was removed in vacuo. Mycelia wasagain extracted with n-hexane and concentrated in vacuo to obtain crudesubstance I, 1.9 g. This was charged on the column of silica gel (95 g,Merck Art. 7734) packed with n-hexane, washed with mixture ofn-hexane-ethyl acetate (10:1), and eluated with n-hexane-ethyl acetate(10:2). The eluate was concentrated in vacuo to obtain crude substanceII, 22.7 mg. The crude substance II was charged on a silica gel thinlayer plates (Merck Art, 5744) and developed with n-hexane-ethyl acetate(1:1). Active fraction was scratched and eluated withchloroform-methanol (2:1). The eluate was concentrated in vacuo toobtain crude substance III, 13.4 mg. Further the substance III wascharged on Sephadex LH-20 column and eluated with chloroform-methanol(1:2). The eluate was concentrated in vacuo to obtain FT-0554 substanceas white powder 10.0 mg.

EFFECT OF THE INVENTION

[0061] As explained hereinabove, FT-0554 substance of the presentinvention is expected to be a satisfactory and useful medicament fortreatment of parasitosis.

1. A process for producing FT-0554 substance of formula [1]

comprising culturing a microrganism belonging to fungi having activityfor producing FT-0554 substance in a medium, accumulating FT-0554substance in the cultured medium and isolating FT-0554 substance fromsaid culture.
 2. The process according to claim 1, wherein saidmicroorganism is Aspergillus niger FT-0554.
 3. The process according toclaim 1, wherein said microorganism is Aspergillus niger FT-0554 FERMBP-6443.
 4. The process according to claim 1, wherein said microorganismis cultured at a ph of 3-10 and a temperature of 12-45° C.
 5. Theprocess according to claim 1, wherein said microorganism is cultured ata ph of 5-7 and a temperature of 16-36° C.
 6. The process according toclaim 1, wherein said medium is a liquid.
 7. The process according toclaim 1, wherein said medium contains seawater.
 8. The process accordingto claim 1, wherein said microorganism is cultured at a ph of 3-10, atemperature of 12-45° C. and in a medium comprising 50-100% seawater. 9.A microorganism belonging to fungi and having the ability to produceFT-0554.
 10. The microorganism according to claim 9, wherein saidmicroorganism is Aspergillus niger FT-0554.
 11. The microorganismaccording to claim 9, wherein said microorganism is Aspergillus nigerFT-0554 FERM BP-6443.
 12. A biologically pure culture of Aspergillusniger FT-0554 FERM BP-6443.